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首页> 外文期刊>Biochemistry >IDENTIFICATION OF THE ACID/BASE CATALYST IN AGROBACTERIUM FAECALIS BETA-GLUCOSIDASE BY KINETIC ANALYSIS OF MUTANTS
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IDENTIFICATION OF THE ACID/BASE CATALYST IN AGROBACTERIUM FAECALIS BETA-GLUCOSIDASE BY KINETIC ANALYSIS OF MUTANTS

机译：突变体动力学分析鉴定粪肠杆菌β-葡糖苷酶中的酸/碱催化剂

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摘要

The catalytic mechanism of the retaining beta-glucosidase (Abg) from Agrobacterium faecalis involves a double-displacement process in which an alpha-glucosyl-enzyme intermediate is formed with general acid catalytic assistance and then hydrolyzed with general base assistance. Glu170 was identified as an important residue, possibly the acid/base catalyst, on the basis of sequence alignments. This glutamate is conserved in almost all enzymes in family 1 of glycoside hydrolases. Detailed pre-steady-state and steady-state kinetic analyses of the mutant E170G suggested very strongly that Glu170 is the acid/base catalyst. First, k(cat) values were invariant with pH over the range of 5.0-9.0. Secondly, rates of formation of the glycosyl-enzyme, calculated from k(cat)/K-m and k(2), were similar to those of wild-type enzyme for substrates not requiring protonic assistance but dramatically reduced for those needing acid catalysis. Thirdly, addition of azide as a competitive nucleophile increased k(cat) values 100-300-fold for substrates whose rate-limiting step is deglycosylation, yielding beta-glucosyl azide, but had no effect on the wild-type enzyme. Other anionic nucleophiles had similar, but less dramatic effects. Previous results [Gebler, J. C., et al. (1995) 34, 14547-14553] had indicated that Tyr298 is important for catalysis. The kinetic consequences of the mutations in the double mutant E170G-Y298F are additive, resulting in a 10(6)-fold reduction in k(cat) values and allowing the accumulation of a stable (t(1/2) > 7 h) glucosyl-enzyme intermediate. Thus, Glu170 and Tyr298 function independently, and a possible role for Tyr298 in modulating the pK(2) of the catalytic nucleophile is proposed. [References: 31]
机译：来自粪农杆菌的保留β-葡糖苷酶（Abg）的催化机理涉及双重置换过程，其中在一般的酸催化辅助下形成α-葡萄糖基酶中间体，然后在一般的碱辅助下水解。根据序列比对，将Glu170鉴定为重要的残基，可能是酸/碱催化剂。该谷氨酸在糖苷水解酶家族1的几乎所有酶中都是保守的。对突变体E170G进行的详细的稳态前和稳态动力学分析非常有力地表明，Glu170是酸/碱催化剂。首先，k（cat）值在5.0-9.0范围内随pH不变。其次，由k（cat）/ K-m和k（2）计算得出的糖基酶的形成速率与野生型酶的形成速率相似，对于不需要质子辅助但对于需要酸催化的酶而言，其底物大大降低。第三，添加叠氮化物作为竞争性亲核试剂，其限速步骤是去糖基化的底物的k（cat）值增加100-300倍，产生β-葡萄糖基叠氮化物，但对野生型酶没有影响。其他阴离子亲核试剂具有相似但不太显着的作用。先前的结果[Gebler，J.C。，等。 （1995）34，14547-14553]已经表明，Tyr298对于催化是重要的。双突变E170G-Y298F中突变的动力学结果是累加的，导致k（cat）值降低10（6）倍，并允许稳定的积累（t（1/2）> 7 h）葡萄糖基酶中间体。因此，Glu170和Tyr298独立发挥作用，并提出了Tyr298在调节催化亲核试剂的pK（2）中的可能作用。 [参考：31]

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